Study On The Interaction Proteins Between Bemisia Tabaci And Tomato Yellow Leaf Curl Virus

Tomato yellow leaf curl virus?TYLCV?is a representative member of the Geminiviridae.Tomato yellow leaf curl disease?TYLCD?is one of the most serious plant diseases caused by TYLCV in tomato planting areas worldwide.Bemisia tabaci is the only vector of TYLCV.After the virus enters the body of B.tabaci by stylet,it can break through various barriers and finally infect the host plants with the saliva secreted by the salivary glands of the B.tabaci to achieve the spread of the virus.In the disease system of TYLCV-B.tabaci-tomato,the interactions between TYLCV and B.tabaci is important.Therefore,finding the interaction proteins between them is a basic work to explore the transmission mechanism of TYLCV.It has guiding significances for the comprehensive control of B.tabaci and TYLCV.In this study,the interactions between B.tabaci and TYLCV were investigated in two aspects.The first was to use the traditional yeast two-hybrid technology to screen the interactions between TYLCV and B.tabaci.The second was to analyze the transcriptome data of Q-biotype B.tabaci with TYLCV and without TYLCV in our lab to look for the proteins that might be involved in the transmission of TYLCV.In this study,the interactions between B.tabaci and TYLCV were studied using the GAL4 yeast two-hybrid system.Studies have shown that capsid protein?CP?of TYLCV plays a key role in virus transmission,so in this study we first cloned the CP gene.And it was inserted into the bait vector pGBKT7-BD of the GAL4 yeast two-hybrid system,the bait plasmid pGBKT7-CP was successfully constructed.We used the TYLCV CP monoclonal antibody to detect the fusion protein of pGBKT7-CP expressed correctly or not in yeast strain Y2HGold by western blot.The result showed that there is a band between 55 kDa and 43 kDa,the size was close to the predicted fusion protein?52 kDa?indicating that pGBKT7-CP can be expressed correctly in yeast Y2HGold.Y2HGold transformed with pGBKT7-CP?pGBKT7-CP-Y2HGold?was coated on three types of selective culture medium SD/-Trp,SD/-Trp/X-?-Gal and SD/-Trp/X-?-Gal/AbA.pGBKT7-CP-Y2HGold grown normally on SD/-Trp and SD/-Trp/X-?-Gal solid culture medium,and it didn't make X-?-Gal turn blue.pGBKT7-CP-Y2HGold could not grow on SD/-Trp/X-?-Gal/AbA,indicating that pGBKT7-CP could not activate GAL4 yeast two-hybrid system when it existed in Y2HGold alone.At the same time,we compared the growth status of the Y2HGold transformed with pGBKT7-CP and pGBKT7 respectively on SD/-Trp solid medium and liquid medium.The result showed that the colony size of them on the solid medium and the growth rate of them in the liquid medium were basically same,indicating that pGBKT7-CP is not toxic to Y2HGold.Therefore,the pGBKT7-CP meets the criteria for the bait vector required for screening library.The cDNA library plasmid of B.tabaci was constructed into yeast Y187.The library transformation efficiency was 1.7×10⁵cfu/?g,and the library titer was 2.32×10⁸cfu/ml,which met the library construction criteria.The mating procedure was used to screen B.tabaci cDNA library with TYLCV CP as a bait protein.A total of482 blue yeast colonies were grown on SD/-Trp/-Leu/X-?-Gal/AbA selective plates.Then these 482blue clones were cultured on the SD/-Trp/-Leu/-His/-Ade/X-?-Gal/AbA which was the most strictest selective solid medium.Only five yeast colonies could grow normally but did not make the X-?-Gal turn blue,so they didn't meet all the conditions for the positive clones.These five clones might have weak interactions with TYLCV CP.In this study,we analyzed the transcriptome data of Q-biotype B.tabaci with TYLCV and without TYLCV in our laboratory found 62 genes were differential expression.Combined with literature and genomic data of Q-biotype B.tabaci,we cloned the full-length cDNA of arylsulfataseB?ARSB?successfully by gene cloning.Bioinformatic methods were used to analyze the characteristics of ARSB.The maximum likelihood method was used to construct the phylogenetic tree.The expressional levels of ARSB at different developmental stages,different tissues and under TYLCV treatments in B.tabaci were detected by the real-time quantitative PCR?RT-qPCR?.The results showed that the full-length cDNA of ARSB was 1731 bp,which encoded a 576 amino acids protein with a molecular weight of 64.89 kDa.The amino acid sequence of ARSB contained the conserved domain of arylsulfataseB.The amino acid sequence encoded by the ARSB of B.tabaci is clustered with the amino acid sequence encoded by the ARSB of Drosophila?Drosophila serrata,XP₀20800640.1?.The RT-qPCR analyses suggested that the expressional level of ARSB varied remarkably at different developmental stages.The mRNA level in the egg stage was the highest;The mRNA level in the head and thorax was significantly higher than that in the abdomen.The mRNA level of ARSB under TYLCV treatment for 72 h was significantly higher than that without TYLCV treatment.This study indicated that the expressional level of ARSB varied at different developmental stages and different tissues.In addition,it was highly expressed in the B.tabaci with TYLCV,suggesting that the gene may be involved in the response of B.tabaci to TYLCV and the process of transmission.